The present invention relates to a chemotactic cytokine.
The accumulation of eosinophil leukocytes is a characteristic feature of IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema. Eosinophil accumulation also occurs in non-allergic asthma. The immediate bronchoconstriction in response to a provoking stimulus in the asthmatic involves mast cell activation and the release of constrictor mediators. This is followed after several hours in some individuals by a late bronchoconstrictor response associated with a massive influx of eosinophils (1). Repeated provocation results in chronic inflammation in the airways and a marked hyper-responsiveness to constrictor mediators. The magnitude of both the late response and the chronic hyper-responsiveness-correlates with the numbers of eosinophils present in the lung (2,3).
The present invention provides a chemoattractant protein capable of attracting eosinophils and of inducing eosinophil accumulation and/or activation in vitro and in vivo. The chemoattractant protein of the present invention is designated xe2x80x9ceotaxinxe2x80x9d.
Eotaxins are proteins of the Cxe2x80x94C branch of the platelet factor 4 superfamily of chemotactic cytokines. Within the Cxe2x80x94C branch of the platelet factor 4 superfamily of chemotactic cytokines, or chemokines, certain members have the property of attracting eosinophils in vitro and some may induce eosinophil accumulation in vivo. For example, the chemokines RANTES and MIP-1xcex1 attract eosinophils in vitro while MCP-1 and MIP-1xcex2 do not. (xe2x80x9cRANTESxe2x80x9d denotes Regulated upon Activation in Normal T cells Expressed and Secreted, xe2x80x9cMIPxe2x80x9d denotes Macrophage Inflammatory Protein, and xe2x80x9cMCPxe2x80x9d denotes Monocyte Chemo-attractant Protein.)
Naturally-occurring cytokines within the platelet factor 4 superfamily of chemotactic cytokines may have marked inter-species variations in the amino acid sequence of the protein, and in the carbohydrate modifications of the protein, while retaining the same characteristic functional properties. Similar variations in structure may occur in cytokines obtained from different individuals within the same species. Many chemokines within the Cxe2x80x94C branch of the platelet factor 4 superfamily show promiscuity of receptor binding, and the ability of different chemokines to bind to the same receptor is not necessarily dependent on a high degree of homology at the amino acid level. Accordingly, both interspecies and intraspecies variations in protein length, amino acid sequence and carbohydrate modifications are generally to be expected for eotaxins.
The ability to attract eosinophils and to induce eosinophil accumulation and/or activation in vitro and in vivo is a characteristic property of eotaxins. Furthermore, eotaxins generally show substantially no attractive effect for neutrophils in vivo. The eosinophil chemoattractant effect may be an inter-species effect, for example, guinea-pig eotaxin appears to be potent in inducing chemotaxis of human eosinophils in vitro.
An eotaxin may be obtained from an appropriate body fluid, for example, from bronchoalveolar lavage fluid obtained from a human or non-human subject, particularly an allergic subject after an allergen challenge, either experimentally induced or naturally incurred. Other sources of eotaxins are, for example, inflammatory exudate fluids and in vitro cultures of macrophages, lymphocytes, neutrophils, mast cells, airway epithelial cells, connective tissue cells, vascular endothelial cells and eosinophils themselves
For example, an eotaxin may be obtained from a sensitised guinea-pig after allergen challenge. Guinea-pig models are useful as they share many common features with the asthmatic response in man. Eotaxin obtainable from bronchoalveolar lavage fluid of a sensitised guinea-pig by sequential HPLC purification generally has a molecular weight in the range of from 6-16 kDa. (As indicated above, intraspecies molecular weight variations of this order of magnitude are observed in members of the platelet factor 4 superfamily.)
The amino acid sequence of a guinea-pig eotaxin is set out in SEQ. ID. NO. 1, SEQ. ID. NO. 2 and in FIGS. 7 and 8 of the accompanying drawings. Other guinea-pig eotaxins will generally have at least 50% overall homology with the sequence shown in SEQ. ID. NO. 1 (FIG. 7) at the amino acid level. The homology may be at least 60%, for example at least 70%, for example at least 80% with the sequence set out in SEQ. ID. NO. 1 and in FIG. 7.
Percentage homology in the present case is calculated on the basis of amino acids that are identical in corresponding positions in the two sequences under investigation. Conservative substitutions are not taken into account. In the calculation of percentage homology of a putative eotaxin molecule under investigation with the sequence shown in SEQ. ID. NO. 1 (FIG. 7) or with SEQ. ID. NO. 2 (FIG. 8) if the molecule under investigation has a different length from the eotaxin set out in SEQ. ID. NO. 1 or SEQ. ID. NO. 2 (FIG. 7 or FIG. 8), then the calculation is based on the amino acids in the portion of the molecule under investigation that overlaps with the sequence shown in SEQ. ID. NO. 1 (FIG. 7) or SEQ. ID. NO. 2 (FIG. 8). Software packages for the alignment of amino acid sequences and the calculation of homology are available commercially, for example, the xe2x80x9cBestfitxe2x80x9d program available from Genetics Computer Group Sequence Analysis Software, Madison, Wis., U.S.A.
Unless specified otherwise, the specific values of percentage homology between eotaxin and other chemotactic cytokines given in the present specification have been calculated on the basis of the eotaxin set out in SEQ. ID. NO. 1 (FIG. 7).
As indicated above, eotaxins obtainable from species other than guinea-pigs, for example humans, will exhibit inter-species differences of the type demonstrated by other members of the Cxe2x80x94C branch of the platelet factor 4 superfamily of chemokines, for example, differences in protein length, amino acid sequence and carbohydrate modifications. There may, for example, be variations in the C- and/or N-terminal residues. For example, it is expected that the molecular weight of an eotaxin from a species other than guinea-pig will generally fall within the range of from 6 kDa to 16 kDa, but in some cases an eotaxin may have a molecular weight less than 6 kDa or more than 16 kDa.
Similarly, it is expected that in general an eotaxin from a species other than guinea-pig will have at least 40% overall homology with the sequence set out in SEQ. ID. NO. 1 and in FIG. 7 The homology may be at least 50%, for example at least 60%, for example at least 70%, for example at least 80% with the sequence set out in SEQ. ID. NO. 1 and in FIG. 7 There may, however, be eotaxins from species other than guinea-pigs that have less than 40% homology with SEQ. ID. NO. 1 (FIG. 7).
Eotaxins may be identified by any one or more of the characteristics set out above, in particular by their ability to attract and/or actuate eosinophils in vitro and cause their accumulation and/or activation in vivo. A characteristic that assists the identification of a molecule as an eotaxin is the lack of attractive effect on neutrophils.
The present invention provides a method of determining the ability of a substance to induce eosinophil accumulation and/or activation in vivo, that is to say, a method for testing putative eotaxins, which comprises administering the substance, generally intradermally, to a test animal previously treated with labelled, for-example 111In-labelled, eosinophils and subsequently determining the number of labelled eosinophils at a skin site.
One in vitro method that may be used to test a putative eotaxin for the ability to attract and/or activate eosinophils in vitro is the ability of the substance to increase eosinophil intracellular calcium levels. Other general methods for determining chemotactic activity in vitro may be used to test putative eotaxins in vitro.
Confirmation that an eosinophil attractant is an eotaxin may also be made by consideration of sequence homology of that protein with the sequence set out in SEQ. ID. NO. 1 (FIG. 7) and/or with the sequence set in SEQ. ID. NO. 2 (FIG. 8) and/or by consideration of the structural relationship between the protein and the guinea-pig eotaxin.
As mentioned above, RANTES and MIP-1xcex1 are both eosinophil activators. Eotaxin has functional similarities but low structural homology with RANTES and MIP-1xcex1 (31% homology with MIP-1xcex1 at the amino acid level calculated on the basis of SEQ. ID. No. 1 (FIG. 7); 32% homology when calculated on the basis of the overlapping sequences and 26% homology with RANTES at the amino acid level calculated on the basis of SEQ. ID. No. 1 (FIG. 7); 27% homology when calculated on the basis of the overlapping sequences). An eotaxin can be distinguished from RANTES and MIP-1xcex1 not only by the degree of homology but also by the overall differences in sequence and structure.
In addition to full-length eotaxin molecules, the present invention also provides molecules that comprise less than a, full length eotaxin sequence. Such molecules (called xe2x80x9cfragmentsxe2x80x9d herein) may be polypeptides or peptides. For use as an eotaxin substitute, a fragment should retain one or more of the biological activities of the parent molecule.
Eosinophils contain an armoury of chemicals necessary for killing parasites. These chemicals have been implicated in the damage to airway epithelium that occurs in asthma and may relate to the observed changes in airway function (26,27). From our studies we suggest that eotaxins should be considered as important mediators of eosinophil accumulation in vivo. Macrophages, lymphocytes, neutrophils, mast cells, airway epithelial cells, connective tissue cells, vascular endothelial cells and eosinophils themselves are likely candidates as the source of the eosinophil chemoattractant activity generated in the lung. Platelets may also have a role as it has been shown that they can release Cxe2x80x94C chemokines (22). Further, an early platelet deposition may be involved in the subsequent eosinophil accumulation in vivo (28,29) and there is evidence that platelet-activating factor induces the synthesis of an unidentified eosinophil chemoattractant in vivo (30). In this respect, it is of interest that platelet-derived growth factor can induce gene expression of Cxe2x80x94C chemokines in fibroblasts (31). Furthermore, the Cxe2x80x94C chemokines have been implicated in wound healing (18). This may be important in the sub-epithelial basement membrane fibrosis that is a prominent feature of the asthmatic lung. Thus, eotaxins may be involved in both eosinophil accumulation and in chronic structural changes in the lung.
Eotaxins may have an important role in asthma and in other diseases having an inflammatory component where eosinophil accumulation and/or activation is a prominent feature, for example, rhinitis and eczema, especially allergic eczema. Accordingly, agents that inhibit or otherwise hinder the production, release or action of eotaxins have potential as selective therapeutic agents. Such agents and their therapeutic use are part of the present invention.
Such agents include inhibitors that affect the interaction of an eotaxin with eotaxin receptors, for example, by binding to an eotaxin or to an eotaxin receptor. An example of such an inhibitor is receptors themselves which, on administration, can bind an eotaxin and prevent its interaction with naturally-occurring receptors. Such inhibitory receptors may be soluble or insoluble. Receptors which are not involved in cell activation may be bound to, or induced on, cells. Such receptors may also be used to remove endogenous eotaxin.
Further examples of agents that affect the interaction of eotaxins with eotaxin receptors are receptor antagonists, and antibodies, both antibodies directed against (capable of binding with) an eotaxin and antibodies directed against an eotaxin receptor, especially monoclonal antibodies. Any other agent that inhibits or otherwise hinders the binding of an eotaxin to an eotaxin receptor also has therapeutic potential, for example, any other agent that binds to an eotaxin or to an eotaxin receptor. Further agents that have therapeutic potential are those that prevent or reduce activation of eotaxin receptors.
Further agents that inhibit or otherwise hinder the action of eotaxins are those that change the structure of an eotaxin such that it is no longer able to bind to an eotaxin receptor, for example, an enzyme or other agent that degrades eotaxin specifically.
Receptor promiscuity is common among chemokines, so although it is essential that a receptor is capable of binding an eotaxin, the receptor need not necessarily be eotaxin-specific. For example, a receptor may bind MIP-1xcex1, RANTES and/or other eosinophil attractant chemokines as well as an eotaxin.
As indicated above, possibilities for therapeutic intervention include the use of a receptor to which an eotaxin binds, especially a soluble receptor. It may be advantageous to use an eotaxin-specific receptor. Further possibilities for therapeutic intervention include receptor antagonists, for example, based on 3-dimensional structures or the amino acid sequences of eotaxins and/or of eotaxin receptors, and agents found to inhibit eotaxin or other agonists binding to or activating eotaxin receptors. For example, a receptor antagonist or an agonist inhibitor may be a polypeptide in which the sequence of a full-length naturally-occurring eotaxin has been modified, for example, by amino acid substitution, or may be a fragment of an eotaxin (that is to say, a polypeptide or small peptide comprising part of the amino acid sequence of a naturally-occurring eotaxin), or a modified fragment of an eotaxin, for example, modified by amino acid substitution.
Furthermore, knowledge of the sequence and/or structure of eotaxins either alone or in combination with knowledge of the sequence and/or structure of other chemokines that bind to the same receptor(s) as eotaxins, provides useful information for the design of therapeutic agents.
Agents that prevent or inhibit eotaxin synthesis or release may also be used therapeutically. Such agents and their use are also part of the present invention.
All inhibitors of eotaxin activity, synthesis and release, including soluble receptors, antibodies, antagonists and inhibitors of agonist binding, and their use are part of the present invention.
The present invention accordingly provides an agent that inhibits or otherwise hinders the production, release or action of an eotaxin, especially an agent as described above, for use as a medicament. The invention also provides the use of an agent that inhibits or otherwise hinders the production, release or action of an eotaxin, especially an agent as described above, in the manufacture of a medicament for the treatment of asthma or another disease having an inflammatory component, particularly with accumulation of eosinophils, for example, rhinitis or eczema, especially allergic eczema.
The use of the structural and sequence information relating to eotaxins in the design of therapeutically and diagnostically useful agents, for example, in computer-aided design based on the three dimensional structure of eotaxins is part of the present invention.
Putative inhibitors of eotaxin activity may be screened using in vivo and in vitro assays based on inhibition of chemoattraction and/or accumulation and/or activation of eosinophils by eotaxins. Some general methods for testing the activity of a compound for an inhibitory effect on the activity of a chemoattractant cytokine in vitro are known. Such assays may be used to determine the inhibitory action of a putative inhibitor on in vitro effects induced in eosinophils by eotaxins.
Assays that are suitable for screening putative eotaxin inhibitors include, for example, inhibition in vitro of elevation of intracellular calcium levels induced in cells by eotaxin. The method of the present invention for determining the ability of a substance to induce eosinophil accumulation and/or activation in vivo, that is to say, a method for testing putative eotaxins, may also be used to determine the ability of a substance to inhibit eosinophil accumulation and/or activation induced in vivo by an eotaxin: an animal is pretreated with labelled eosinophils, an eotaxin and a putative inhibitor are administered, and the number of labelled eosinophils at a skin site are determined subsequently. The eotaxin is generally administered intradermally, and the putative inhibitor may be administered by the same route or by a different route, for example systemically.
Examples of in vitro and in vivo assays both for the determination of eotaxin activity and for the determination of eotaxin inhibitory activity are described herein. For example, Example 1 gives a detailed protocol for the in vivo assay of the present invention, and Example 4 gives detailed protocols of various assays. The assays described herein may be used as such, or may be modified as required. Assays may be used alone or in combination to establish eotaxin and eotaxin-inhibitory activity. A putative inhibitors may be any of the types of molecules described above, including receptors, for example, soluble receptors, antibodies, and antagonists and inhibitors of agonist binding. Methods for testing putative inhibitors of eotaxins are also part of the present invention.
A further aspect of the present invention is a pharmaceutical preparation comprising, as active ingredient, an agent that inhibits or otherwise hinders the production, release or action of an eotaxin, in admixture or in conjunction with a pharmaceutically suitable carrier. Such agents are described above and include, for example, an inhibitor of eotaxin synthesis or release, a soluble eotaxin receptor, an eotaxin receptor antagonist or an inhibitor of an eotaxin receptor agonist, an antibody against eotaxin or an antibody against an eotaxin receptor.
The invention further provides a method of treating asthma and other inflammatory diseases, comprising the administration of an effective amount of an agent that inhibits or otherwise hinders the production, release or action of an eotaxin. The agent may be as described above, for example, an inhibitor of eotaxin synthesis or release, a soluble eotaxin receptor, an eotaxin receptor antagonist or an inhibitor of an eotaxin receptor agonist, or an antibody against eotaxin or against an eotaxin receptor.
The present invention also provides assays for eotaxins and for anti-eotaxin antibodies, especially immunoassays and in particular ELISAs (enzyme-linked immunosorbent assays). The invention provides, for example, an immunoassay for an antigen, characterised in that the antigen is an eotaxin, and also provides an immunoassay for an antibody, characterised in that the antibody is an anti-eotaxin antibody. The invention also provides assays for eotaxins that are analogous to immunoassays for eotaxins but that use a specific-binding partner other than an antibody. In such specific-binding partner assays an eotaxin receptor may be used instead of an anti-eotaxin antibody.
In an immunoassay, an anti-eotaxin antibody may, for example, be coated on a solid surface to enable capture and hence detection of eotaxin An anti-eotaxin antibody may be used in an assay for the detection of antibodies to eotaxin, for example, in a competitive antibody assay. A labelled eotaxin or a derivative thereof, for example, a recombinant eotaxin or a synthetic peptide comprising part of the amino acid sequence of an eotaxin may be used in a competitive antigen assay for eotaxin or may be used to coat a solid surface in a capture assay for antibodies to eotaxin. The many different types of assay format are well described in the literature of the art, see for example xe2x80x9cELISA and other Solid Phase Immunoassays, Theoretical and Practical Aspectsxe2x80x9d Eds Kemeny D. M. and Challacombe S. J., John Wiley, 1988. (36). Assays using an eotaxin receptor instead of an anti-eotaxin antibody may be carried out analogously.
The present invention provides a process for the production of an eotaxin, which comprises obtaining bronchoalveolar lavage fluid obtained from a human or non-human animal challenged with a provoking stimulus, for example, from a human suffering from allergic or non-allergic asthma, or other lung disease, or a guinea-pig sensitised with a foreign protein, and isolating a fraction showing eosinophil chemoattractant activity. One method of isolating an eotaxin-containing fraction of bronchoalveolar lavage fluid is sequential cation exchange, size exclusion and reversed phase HPLC systems. The desired fraction generally contains a polypeptide having a molecular weight in the range from 6-16 kDa. Purity may be verified by SDS-PAGE. If desired, the authenticity of the substance obtained may be determined by comparison of the amino acid sequence thereof with the amino acid sequence set out in SEQ. ID. NO. 1 or SEQ. ID. NO. 2 (FIG. 7 or FIG. 8).
Eotaxins may be obtained according to the above procedure from other sources, for example, from inflammatory exudate fluids, or from in vitro cultures of macrophages, lymphocytes, neutrophils, mast cells, airway epithelial cells, connective tissue cells, vascular endothelial cells and eosinophils themselves.
Alternatively, a full-length eotaxin, or a part (fragment) of an eotaxin, for example, a polypeptide or peptide fragment, may be produced by chemical synthesis, for example, according to the Merryfield technique. A further method for producing a full-length eotaxin or a part thereof is by recombinant DNA technology. All methods of producing eotaxin are part of the present invention.
To produce a full-length eotaxin polypeptide or a polypeptide (or peptide) fragment by recombinant DNA technology, a nucleic acid sequence encoding the polypeptide is inserted into an expression vector under the control of appropriate control sequences. A recombinant polypeptide may then be expressed using a prokaryotic expression system, for example, in E. coli, or using a eukaryotic cell system, in which case the resulting polypeptide may be glycosylated. Such techniques are standard see, for example Sambrook, J., Fritisch, E. F. and Maniatis T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989 (37).
A nucleic acid encoding all or part of an eotaxin polypeptide may be obtained by screening a library prepared from suitable cells, for example, cells from allergen-challenged guinea-pig or human lung. Screening may be carried out using a probe comprising sequences characteristic of an eotaxin, in particular, sequences that distinguish the eotaxin from other related cytokines, for example, RANTES and MIP-1xcex1. It may be preferable to use a long probe, for example, a probe comprising a nucleic acid sequence encoding a full-length eotaxin polypeptide.
Alternatively, the polypeptide sequence of an eotaxin may be used to design oligonucleotide primers, for example, degenerate primers. Primers may, for example, comprise bases less specific in their interaction than the naturally-occurring bases, for example, inosine may be used. Examples of primers are the sense sequence
5xe2x80x2 TGC TGT TTC CGI GTI ACT AAC AAA (SEQ. ID. NO. 3) based on the amino acid sequence CCFRVTNK, and the anti-sense sequence
5xe2x80x2 CAT CTT GTC IGG CTT TAT TTC (SEQ. ID. NO. 4)
based on the amino acid sequence EIKPDKM. Such primers may be used for amplification of reverse transcribed mRNA by the polymerase chain reaction. This provides cDNA probes for screening libraries, for example, as described above, to isolate full length eotaxin clones. Primers may include codons chosen on the basis of known species preference.
As indicated above, derivatives of naturally occurring eotaxins are also part of the present invention. Such derivatives include polypeptides that have one or more of the following modifications relative to a naturally-occurring eotaxin:
(i) elongation or shortening at the C-terminus;
(ii) elongation or shortening at the N-terminus;
(iii) deletion and/or insertion of internal sequences;
(iv) amino acid substitutions, for example at the C- and/or N-terminus; and
(v) a different pattern of glycosylation. (There is, for example, a potential O-glycosylation site at residue 70.)
Such derivatives may function as agonists for structure/activity relationship studies or as receptor antagonists.
Anti-eotaxin antibodies and anti-eotaxin receptor-antibodies, both polyclonal and monoclonal, may be produced according to standard techniques, for example, Kohler and Milstein (38). A full-length eotaxin may be used as antigen, or it may be preferred to use a fragment of an eotaxin in order to produce an antibody to a specific antigenic determinant A naturally occurring eotaxin may used as antigen. Alternatively, a recombinant or synthetic eotaxin or eotaxin polypeptide or peptide may be used.